S248: THE ENDOSOMAL ADAPTOR PROTEIN MYCT1 CONTROLS ENVIRONMENTAL SENSING IN HUMAN HSCS
Authors: J. Aguade-Gorgorio, Y. Jami-alahmadi, M. Kardouh, I. Fares, V. Calvanese, H. Johnson, M. Magnusson, J. Shin, H. Goodridge, J. Wohlschlegel, H. Mikkola
Journal: HemaSphere (2022)

Background: Hematopoietic stem cells (HSC) integrate diverse environmental cues to maintain the balance between quiescence, self-renewal and differentiation throughout life. However, the molecular programs governing HSC stemness become dysregulated during culture, compromising HSC self-renewal and engraftment ability. Despite recent advances in optimizing culture conditions for human HSC, our ability to expand or modify functional human HSCs in culture for therapeutic use is still limited. Our data uncovered MYCT1 (Myc target 1) as a critical human HSC regulator that is selectively expressed in undifferentiated HSPCs and endothelial cells (EC) but becomes drastically downregulated during culture, concomitantly with loss of engraftment potential. Aims: We aimed to understand the molecular programs controlled by MYCT1 that are essential for maintaining human HSC function. Methods: We analyzed multiple RNAseq gene expression data sets of cultured and uncultured human HSPCs from developmental and postnatal hematopoietic tissues to identify genes associated with self-renewing HSCs. To assess the importance of MYCT1 expression in human HSC function we transduced fetal liver (FL) and cord blood (CB) HSPCs (CD34+CD38-CD90+ and GPI80+ in FL) with lentiviral shRNA vectors. We quantified the expansion of control or knockdown (KD) HSPCs at distinct time points, and determined engraftment ability at 6, 12, and 24 weeks after transplanting equal numbers of sorted HSPCs into conditioned immunodeficient mice. To define MYCT1 localization and interactome we performed subcellular fractionation, immunofluorescence, and immunoprecipitation coupled with high-sensitivity mass spectrometry in endothelial cells and the HSC-like KG1 cell line. We assessed endocytosis by quantifying the internalization of fluorescently labeled dextran and transferrin by FACS in human HSPCs and ECs after MYCT1 KD or overexpression (OE). To determine the signaling and cellular consequences of MYCT1 loss we performed phospho-proteomics, western blot, and single cell RNAseq in ECs and/or human HSPCs. HSPC proliferation was quantified after MYCT1 KD or OE by CFSE dilution assay and monitoring of single cell divisions for 5 days. Results: MYCT1 KD experiments revealed that MYCT1 is critical for human HSPC ex vivo expansion and engraftment after transplantation. We determined that MYCT1 is a membrane-associated protein localized in endosomes, where it interacts with vesicle trafficking components and signaling receptors with critical functions in HSC biology. MYCT1 KD in human ECs and HSPCs led to hyperactivation of endocytosis, a crucial regulatory step determining the responsiveness to extracellular cues and governing cell fate decisions. MYCT1 KD then led to widespread dysregulation of signaling pathways, including a hyperactivation Akt, and cellular functions, including defective proliferation, whereas MYCT1 overexpression had the opposite effect Summary/Conclusion: Our data suggest that MYCT1 governs human HSC stemness and fate decisions by controlling the sensing of extracellular signals and their downstream signaling through endocytosis. As MYCT1 expression is downregulated in cultured HSPC, this work suggests that the inability to properly sense microenvironmental signals in cultured HSCs is a key mechanism contributing to culture-associated HSC dysfunction that will need to be overcome to improve transplantability of ex vivo expanded human HSCs.